Liquid chromatography (LC) is an analytical technique in which an analyte or sample is examined. A LC system typically has one or more columns that are packed with a stationary phase material. Generally, the term “column” refers to columns, cartridges, capillaries and the like for performing separations of a chromatographic nature. Columns are typically packed or loaded with a stationary phase. This stationary phase can be particulate or beadlike or a porousmonolith or a substantially inert material. For the purpose of the present disclosure, the term “column” also refers to capillaries which are not packed or loaded with a stationary phase but rely on the surface area of the inner capillary wall to effect separations.
A mobile phase material or solution mixed with the analyte is pumped into the column and the stationary phase material separates and isolates the analyte. The mobile phase material may comprise any fluid, such as, liquids, gases, supercritical fluids or mixtures thereof. Often the analyte elutes directly from the column to an inline detector to generate a chromatogram.
Typically, the analyte is only available in minute quantities so that extreme care must be taken not to waste even the smallest amount. Consequently, LC systems are designed to operate on minute samples or nano-flows with columns that are nano-sized capillaries such as described in U.S. Pat. No. 6,299,767, which is incorporated herein by reference.
Several systems have been developed to accurately and efficiently deliver the mobile phase material or gradient. The delivery of the gradient is often under demanding constraints such as high pressure, e.g., high pressure liquid chromatography (HPLC) systems. For example, U.S. Pat. Nos. 6,858,435 and 6,610,201 describe various techniques in which the solvent composition is formed and delivered. First in, first out (FIFO) is a commonly used technique for forming and delivering the gradient.
Despite the advances noted above, many inherent limitations remain in nano-flow LC systems. Very commonly the gradient is formed for real-time, immediate consumption by the column. As a run may take an hour, this increases the difficulty of maintaining the proper mixture of the gradient under challenging circumstances. In particular, nano-flow LC systems utilize nano-scale flow transducers.
However well-suited to the particular application, nano-scale transducers have a limited dynamic range and are detrimentally subject to thermal effects. Formation of the gradient over a typical period, such as an hour, during which the temperature can fluctuate results in a poor mixture consistency because of the thermal drift of the transducer. Further, on a nano-scale where the transducers may only hold 2 μL of fluid, the high pressure compression of the fluids generates erroneous transducer readings which spoil the mixture. High pressure operation is also at the edge of the tranducer's dynamic range where noise levels can become unacceptable. In short, limited range, noise and thermal drift associated with the transducers are obstacles to meeting the desired performance.
For example, a commercially available nano-flow thermal anemometry type transducer, having a calibration range of 0 to 5 uL/min, operating at an elution flow rate of 250 nL/min, requires repeatable calibration performance of the organic pump down to 7.5 nL/min, for an initial chromatographic starting composition of 3%. This is not an easy performance specification to achieve because the noise level of the device can be as high as +/−5 nL/min, and the thermal sensitivity to noise without thermal compensation can be in excess of +/−4 nL/min per degree Celsius. Thus, a one degree change of the instrument temperature can result in almost a 50% composition error. In an effort to remedy this error, the typical commercially available direct nano-flow LC system employs an external thermal compensation scheme with a temperature sensing element to compensate the flow transducer. Such attempts at compensating for the affects of the thermal sensitivity greatly increase the complexity of the LC system without addressing the underlying problem.
Often, the bulk flow to the LC system is changed during starting and stopping flow, changing flow rates and transitioning flows between operations. During bulk or solvent flow change, it is difficult to maintain the desired composition mixture across the mixing point or node. For example, when the steady-state back pressure of the LC system is very high (e.g., greater than 5,000 psi), the two fluid streams (e.g., solvent and analyte) have significantly different fluid properties (i.e., aqueous and organic solvents) before the mixing node. Due to inherent limitations, such as flow transducers with relatively slow response times (i.e., time constants of several seconds), the necessary feedback is not provided quickly enough to allow the system to be responsive and maintain the desired composition mixture.
Rapid pressure changes from say 10,000 PSI to 200 PSI can cause critical and expensive components like the column to be destroyed. At the very least, the cycles of compression and decompression create apparent flow when none is actually occurring. Also, the compressibility changes of fluid create false readings because the transducers are inherently sensitive to such rapid fluid density changes as the back pressure swings across the typically large range of pressure, and to some extent, the adiabatic heating effect of the fluids. Thus, LC systems that regulate flow between multiple solvent pumps using feed back control cannot maintain accurate measured flow due to the effects of the rapid compressibility changes and heating on the nano-flow transducers.
Another disadvantage of prior art LC systems is cross-flow and back-flow contamination of the flow transducers across the mixing node, which results in contamination of the LC system fluid stream and a temporary loss of flow calibration to the transducers. During rapid decompression or during the normal chromatographic delivery of the gradient, as the viscosity of the fluid drops (i.e., higher organic), prior art LC systems require flow to be reversed back through the mixing node. To buffer the back-flow contamination of the transducers, the connecting conduits between the flow transducers and the mixing node are sized to accommodate the decompression volume of the mixing tee. However, the additional volume becomes lumped with that of the transducers, further exacerbating the rapid compressibility change problems noted above. Thus, conventional high-pressure mixing systems are very susceptible to cross-contamination across the mixing node at very high pressures.
Further, the cross-contamination across the mixing node can create feedback instability between two flow transducers resulting from the fluidic inter-active coupling. The cross-contamination also increases the loss of maximum operating pressure, which is attributed to the large parasitic pressure drops associated with much larger decoupling restrictors required by high-pressure mixing systems to passively stabilize the control interaction. Previous LC systems have recognized these problems and proposed redundant pumps, complex plumbing schemes and valves to isolate the gradient formation pumps from the high-pressure portion of the LC system. Such costly and complex solutions are not only undesirable but the problems remain.
Still further, some LC applications require changing the operational flow rate for a particular choice of column during an injection run, e.g., sample trapping and 2-D chromatography. The flow rate must be started and stopped between selection of each column. Prior art systems have employed some means of valve switching at essentially a no-flow condition to accomplish such flow rate changes. The valve switching components deter from the reliability of the LC system while increasing the expense and complexity in an unfavorable manner.
Other systems have also been developed in an effort to increase the sensitivity and/or collect more data from samples. For example, U.S. Pat. No. 6,858,435 discloses LC analysis systems that make use of a variable flow or peak parking to overcome the difficulty a detector may have in adequately sensing the various species with the sample liquid. When the LC analysis system detects a peak of interest, the LC analysis system controls a micro-switching valve to rapidly reduce the elution flow rate (i.e., reduction in flow by 20 to 50 times). As a result, the elution time of the column-separated compounds is extended to enhance detection. After analysis, the LC analysis system restores the normal elution flow rate. Again, the employment of additional components to accomplish peak parking unfavorably increases the expense and complexity of the system.
Another method to increase the efficiency of LC analysis is to utilize microscale or nanoscale flow rates such as 0.025 to 100 ul/min flow rates. By using such flow rates, the LC analysis system can produce ultra high sensitivity analysis. However, gradient delay and dispersion become problematic. Further, sample loading time and thereby the whole runtime become undesirably long.
As can be seen from the discussion above, closed-loop feedback mechanisms have been developed for LC analysis systems. However, there is a need for still better control and prior art systems do not use feed-forward open-loop mechanisms. Feed-forward is an approach to reacting to changes in a system to minimize or prevent error.